Method for the purification of prostaglandins

ABSTRACT

The present invention provides a method for the purification of a prostaglandin by supercritical fluid chromatography, said method comprising the use of a stationary phase and a mobile phase comprising carbon dioxide, provided that when the stationary phase is unmodified silica gel, the prostaglandin is not luprostiol. The invention also provides prostaglandins obtainable by the method.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No. 13/953,236, filed Jul. 29, 2013, which is a continuation of U.S. patent application Ser. No. 12/820,610, filed Jun. 22, 2010, which is now U.S. Pat. No. 8,519,178, issued Aug. 27, 2013, and claims priority to U.S. Provisional Application No. 61/219,166, filed Jun. 22, 2009, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

FIELD OF THE INVENTION

The present invention provides a method for the purification of prostaglandins. In particular, the present invention provides a method for the purification of prostaglandins by supercritical fluid chromatography (SFC).

BACKGROUND OF THE RELATED ART

Prostaglandins are active pharmaceutical ingredients (APIs) and isomerically and chemically pure prostaglandins are required for formulation into drug products. However, the purification of prostaglandins is challenging due to the similar chemical properties of many prostaglandin isomers, as well as their related impurities.

G. H. Brunner et al. (Supercritical Fluids, 653-668, E. Kiran and J. M. H. Levelt Sengers (eds.), Kluwer Academic Publishers, 1994) describes the preparative supercritical fluid chromatography (SFC) separation of Reprodin isomers (i.e. luprostiol isomers). However, the isomers are poorly separated and the purity of the heart cut fraction is only 80%. This method therefore cannot be used to obtain a commercial product as the purity does not meet ICH quality. The authors acknowledge that SFC is disadvantageous in comparison to liquid chromatography.

BRIEF SUMMARY OF THE INVENTION

The present inventors, however, have overcome the problems associated with the prior art procedure to provide an alternative process for the purification of prostaglandins using SFC.

Accordingly, the present invention provides a method for the purification of a prostaglandin by supercritical fluid chromatography, said method comprising the use of a stationary phase and a mobile phase comprising carbon dioxide, provided that when the stationary phase is unmodified silica gel, the prostaglandin is not luprostiol. For example, the invention includes a method for purifying a crude prostaglandin, comprising injecting the crude prostaglandin onto a column comprising a stationary phase, eluting the crude prostaglandin through the column using a mobile phase comprising supercritical carbon dioxide, and collecting a fraction comprising a purified prostaglandin.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a chromatogram of a SFC purification injection of crude latanoprost, obtained as described in Example 1.

FIG. 2 sets out the structures of latanoprost isomers.

FIG. 3 illustrates a chromatogram of a SFC purification injection of crude latanoprost, obtained as described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

By “purification”, it is meant the method produces a prostaglandin which is chemically and/or isomerically pure. A chemically pure compound is one where the compound is essentially free from related compounds, chemical starting materials, chemical intermediates and chemical degradants. An isomerically pure compound is one where the compound is essentially free from known related compounds of the same chemical molecular formula that are different in chemical structure.

In one embodiment, the purified prostaglandin is at least about 99% chemically pure, preferably at least about 99.5% chemically pure and more preferably at least about 99.8% chemically pure.

In another embodiment, the purified prostaglandin is at least 99% isomerically pure, preferably at least about 99.5% isomerically pure and more preferably at least about 99.9% isomerically pure.

The method of the present invention may be utilised to purify a prostaglandin on an analytical or preparative scale. By “analytical”, we mean a scale of providing at least about 0.1 mg of purified prostaglandin, preferably about 1 mg of purified prostaglandin, in a reasonable timeframe, i.e. less than a day. By “preparative”, we mean a scale of providing at least about 1 mg of purified prostaglandin, preferably about 0.1 g of purified prostaglandin and most preferably about 1 g of purified prostaglandin, in a reasonable timeframe, i.e. less than a day.

Preferably, the prostaglandin is a compound of formula (I), a compound of formula (II), a compound of formula (III) or a compound of formula (IV):

wherein,

V is C or O; W is

X is CONR₁₂R₁₃ or CO₂R₁₂; Y is

Z is C₁₋₂₀-alkyl, C₂₋₂₀-alkynyl, —O—(C₆₋₂₀-aryl) or —(C₁₋₂₀-alkyl)-(C₆₋₂₀-aryl), wherein the aryl group is optionally substituted with one to three substituents selected from the group consisting of C₁₋₂₀-alkyl, halo and C(halo)₃; R₁ and R₂ are independently H or OH, or R₁ and R₂ together form

R₃ and R₄ are independently H or OH; R₅ and R₆ are independently H or OH, or R₅ and R₆ together form

R₇ and R₈ are independently H, OH, halo or C₁₋₂₀-alkyl; R₉ and R₁₀ are independently H or C₁₋₂₀-alkyl, wherein the alkyl group is optionally substituted with one or more substituents selected from CONR₁₂R₁₃, CO₂R₁₂ or CO₂ ⁻M⁺; R₁₁ is C₁₋₂₀-alkyl or —O—(C₁₋₂₀-alkyl), wherein the alkyl group is optionally substituted with one or more substituents selected from CONR₁₂R₁₃, CO₂R₁₂ or CO₂ ⁻M⁺; R₁₂ and R₁₃ are independently selected from the group consisting of H and C₁₋₂₀ alkyl; M⁺ is a counter cation; and Halo is fluorine, chlorine, bromine or iodine. When V═C, it is understood that the carbon atom bears two hydrogen atoms, one or both of which may be substituted with a substituent group which may be independently selected from halo and C₁₋₂₀-alkyl, for example.

“Alkyl” refers to linear, branched or cyclic saturated hydrocarbon structures having, unless otherwise indicated, 1 to 20 carbon atoms, more preferably 1 to 15 carbon atoms and most preferably 1 to 10 carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, i-butyl, t-butyl, n-pentyl, n-hexyl and cyclohexyl. When an alkyl group having a specific number of carbons atoms is named, it is intended that all geometric isomers of that alkyl group are encompassed. For example, “butyl” includes n-butyl, i-butyl, t-butyl and cyclobutyl.

“Alkynyl” refers to linear or branched hydrocarbon structures having at least one

group and, unless otherwise indicated, 2 to 20 carbon atoms, more preferably 2 to 15 carbon atoms and most preferably 2 to 10 carbon atoms. Examples of alkynyl groups are ethynyl, propynyl, n-butynyl, isobutynyl and hexynyl. When an alkynyl group having a specific number of carbon atoms is named, it is intended that all geometric isomers of that alkynyl group are encompassed. For example “butynyl” includes n-butynyl and isobutynyl.

“Aryl” refers to an aromatic hydrocarbon structure having, unless otherwise indicated, 6 to 20 carbon atoms, more preferably 6 to 15 carbon atoms and most preferably 6 to 10 carbon atoms. Examples of aryl groups are phenyl and naphthyl.

The term “halo” whether alone or as part of another group refers to a halogen, for example, a fluorine, chlorine, bromine or iodine atom.

M⁺ is a counter cation of —CO₂ ⁻ i.e. —CO₂ ⁻M⁺ is a carboxylic acid salt and preferably a pharmaceutically acceptable carboxylic acid salt. Preferably M⁺ is a metal ion e.g. an alkali metal ion, such as K⁺ or Na⁺.

Preferably, W is

X is preferably CONH(C₁₋₁₀-alkyl), CO₂(C₁₋₁₀-alkyl) or CO₂H. More preferably, X is selected from the group consisting of CONHEt, CO₂Me, CO₂ ^(i)Pr and CO₂H.

Preferably, Z is C₁₋₁₀-alkyl, C₂₋₁₀-alkynyl, —O—(C₆₋₁₀-aryl) or —(C₁₋₁₀-alkyl)-(C₆₋₁₀-aryl), wherein the aryl group is optionally substituted with one to three substituents selected from the group consisting of Cl and —CF₃. More preferably, Z is —(CH₂)₅CH₃, —(CH₂)₃CH₃, —CH₂—Ph,

R₁ and R₂ may independently be H or OH. Preferably, when one of R₁ and R₂ is H, the other of R₁ and R₂ is OH. More preferably, when one of R₁ and R₂ is H and the other of R₁ and R₂ is OH, R₁, R₂ and the carbon atom to which they are attached have the following stereochemistry:

R₃ and R₄ are independently H or OH. Preferably, when one of R₃ and R₄ is H, the other of R₃ and R₄ is OH. More preferably, R₃, R₄ and the carbon atom to which they are attached have the following stereochemistry:

R₅ and R₆ may independently be H or OH. In one embodiment, when one of R₅ and R₆ is H, the other of R₅ and R₆ is OH. More preferably, when one of R₅ and R₆ is H and the other of R₅ and R₆ is OH, R₅, R₆ and the carbon atom to which they are attached have the following stereochemistry:

When R₅ and R₆ together form

and one of R₃ and R₄ is OH and the other of R₃ and R₄ is H, it is possible for a tautomer to be produced i.e. a hemiacetal. It is envisaged that such tautomers are encompassed with the scope of the present invention. A tautomeric equilibrium is exemplified by the prostaglandin lubiprostone:

In another embodiment, R₅ and R₆ are both H.

Preferably, R₇ and R₈ are independently selected from the group consisting of H, OH, F or CH₃. In one embodiment, R₇ and R₈ are both H. In another embodiment, R₇ and R₈ are both F. In yet another embodiment, one of R₇ and R₈ is CH₃ and the other of R₇ and R₈ is H or OH.

Preferably, R₉ and R₁₀ are independently H or C₁₋₁₀-alkyl, wherein the alkyl group is optionally substituted with one or more substituents selected from CO₂H or CO₂ ⁻M⁺. More preferably, one of R₉ and R₁₀ is H and the other of R₉ and R₁₀ is —(CH₂)₃CO₂H or —(CH₂)₃CO₂ ⁻Na⁺.

Preferably, R₁₁ is C₁₋₁₀-alkyl or —O—(C₁₋₁₀-alkyl), wherein the alkyl group is optionally substituted with one or more substituents selected from CO₂H or CO₂ ⁻M⁺. More preferably, R₁₁ is —(CH₂)₃CO₂H or —O—CH₂—CO₂H.

In a preferred embodiment, the prostaglandin is selected from the group consisting of:

Preferably, the prostaglandin is latanoprost.

Alternatively, if the stationary phase is other than unmodified silica gel, the prostaglandin can be luprostiol:

In one embodiment, the stationary phase is a chiral stationary phase. Preferably, the chiral stationary phase is a derivatised amylose or cellulose polymer or other polysaccharide which is coated or immobilized on silica. More preferably, the chiral stationary phase is selected from the group consisting of Chiralcel OD-H, ChiralPak AS-H, ChiralPak IC, ChiralPak AD-H, Chiralcel OJ-H and Chiralcel OK (products available from Chiral Technologies Inc. and Daicel Chemical Industries, Ltd.). More preferably, the chiral stationary phase is ChiralPak AD-H. For example, the amylase or cellulose polymer may be derivatised with one or more carbamate groups, especially aryl-containing carbamate groups such as 3,5-di methylphenylcarbamate, (S)-alpha-methylbenzylcarbamate, 4-chlorophenyl carbamate, 4-methylphenylcarbamate, phenyl carbamate, 3-chloro-4-methylphenylcarbamate, 5-chloro-2-methylphenylcarbamate or the like, and/or one or more ester groups, such as acetate, benzoate (e.g., 4-methyl benzoate), cinnamate, or the like.

In another embodiment, the stationary phase is a non-chiral stationary phase. Preferably, the non-chiral stationary phase is selected from the group consisting of Princeton Diol, 4-ethyl pyridine, 2-ethyl pyridine and pyridine urea.

Preferably, the mobile phase further comprises at least one modifier. The modifier can be any suitable liquid solvent. A suitable modifier may be selected from the group consisting of at least one alcohol, acetonitrile, ethyl acetate, methylene chloride and a combination thereof. Preferably, the at least one alcohol is selected from the group consisting of methanol, ethanol, propanol, isopropanol and a combination thereof. It is desirable that the modifier is compatible with the stationary phase. For example, ethyl acetate and methylene chloride cannot be used with a ChiralPak AD column as they will destroy the column.

Carbon dioxide is easily removed and so, the purified prostaglandin can be provided as a solution with the modifier as a solvent. It may therefore be desirable to select a modifier in which the prostaglandin is soluble.

Suitably, the at least one modifier is present in a quantity from (i.e., of at least) about 1% v/v or about 1% w/w to the supercritical carbon dioxide. More preferably, the at least one modifier is present in a quantity from (i.e., of at least) about 5% v/v or about 5% w/w to the supercritical carbon dioxide. The ratio of modifier to carbon dioxide can be varied during the chromatographic process.

Suitable chromatographic apparatus is well known to the skilled person. It is preferred to use an apparatus that is suitable for Supercritical Fluid Chromatography such as the Thar Investigator SFC or Novasep Supersep 20/30 SFC. The crude feed containing the crude prostaglandin is periodically injected into the apparatus wherein the mobile phase flows through the stationary phase which is located in a column. After detection at the column outlet, the purified fractions of the feed are directed to different traps. The carbon dioxide is removed from the purified fractions and is preferably recycled. Detection at the column outlet can be conducted by measuring UV absorption at an appropriate wavelength.

On an analytical scale, the column diameter is suitably from about 2 mm to about 7 mm, preferably about 4.6 mm. The column length is suitably from about 5 cm to about 50 cm, preferably about 25 cm.

On a preparative scale, the column diameter is suitably from about 10 mm to about 200 mm, preferably about 21 mm. The column length is suitably from about 5 cm to about 50 cm, preferably about 25 cm.

The process is suitably carried out at temperatures from about 5° C. to about 45° C., preferably about 25° C. to about 35° C., and at elevated pressures e.g. from about 80 bar to 300 bar, preferably about 100 bar to 150 bar. Typical flow rates depend upon the diameter of the column and may vary from e.g. 1 mL/min to about 5 kg/min.

In another aspect, the present invention provides a prostaglandin obtainable by a method as described above.

The following Examples are illustrative but not limiting of the invention.

Example 1 Analytical Scale

SFC Preparatory Parameters:

-   -   Column: Chiral Technologies Chiralpak AD-H, 4.6×250 mm     -   Column Temperature: 35° C.     -   System Back Pressure: 150 Bar     -   Eluent: Carbon Dioxide (CO₂) with an alcoholic modifier, Ethanol     -   Total Flow Rate: 3 mL/min     -   Gradient Co-Solvent Profile: Initial conditions=5% Ethanol: 95%         CO₂, linearly increase to 45% Ethanol: 55% CO₂ in 15 minutes,         maintain 45% Ethanol: 55% CO₂ for 2 minutes, ramp back to         initial conditions and equilibrate for 3 minutes prior to next         injection     -   Detection: UV at 210 nm     -   Sample Preparation: Prepare a 0.1 g/mL solution in Ethanol and         thoroughly mix the feed solution to completely dissolve the         Latanoprost     -   Sample Loading: 5 mg (50 μL of 0.1 g/mL crude solution in         Ethanol)     -   Latanoprost Collection: Collect the Latanoprost peak (heart cut)         from approximately 2% above the baseline from the detected         Latanoprost peak front (at ˜9 minutes in retention time) to the         Latanoprost detected peak tail (at ˜10 minutes in retention         time).     -   The chromatogram of a SFC purification injection of Latanoprost         Crude is provided in FIG. 1.     -   Obtained Results using Ethanol as the Co-Solvent and AD-H         Stationary Phase:

Chromatographic Purity - Reported as Measured % Peak Area Latanoprost Isomeric Content* Iso- C11- C15R- C15S- C15S- Sample Chemical meric beta trans cis trans Crude Feed 95.87% 90.02% 1.11% 3.88% 3.04% 0.05% (0.01 g/mL) Heart Cut 99.95% 99.92% — — 0.08% — (5 mg Loading) *The structures of the latanoprost isomers are set out in FIG. 2.

Example 2 Preparative Scale

SFC Preparatory Parameters:

-   -   Column: Chiral Technologies Chiralpak AD-H, 21×250 mm     -   Column Temperature: 25° C.     -   System Back Pressure: 100 Bar     -   Eluent: Carbon Dioxide (CO₂) with an alcoholic Co-solvent         modifier (specifically Ethanol: Methanol (4:1))     -   Total Flow Rate: 50 g/min     -   Co-Solvent Profile: Initial conditions=15% Co-solvent: 85% CO₂,         for 360 seconds, Step to 40% Co-solvent: 60% CO₂ at 361 seconds         until 720 seconds, step back to initial conditions at 721         seconds until 900 seconds     -   Detection: UV at 220 nm     -   Crude Solution Preparation: Prepare a 0.3 g/mL solution in         Ethanol and thoroughly mix the feed solution to completely         dissolve the Latanoprost     -   Sample Loading: ˜0.6 mL crude solution in Ethanol     -   Latanoprost Collection: Collect the Latanoprost peak (heart cut)         from approximately 30 seconds after peak start the baseline from         the detected Latanoprost peak front (at ˜410 seconds in         retention time) to approximately 30 seconds prior to the peak         end (at ˜510 seconds in retention time).     -   The chromatogram of a SFC purification injection of Latanoprost         Crude is provided in FIG. 3.     -   Obtained Results using Ethanol as the Co-Solvent and AD-H         Stationary Phase of the collected fractions:

Chromatographic Purity - Reported as Measured % Peak Area Latanoprost Isomeric Content^(§) Iso- C11- C15R- C15S- Sample Chemical meric beta trans C15S-cis trans Crude 96.51% 93.07% 0.70% 2.79% 2.15% 0.06% Feed Purified 99.81%* 99.98% — — 0.02% — Heart Cut *No impurities measured above 0.04% by percent peak area. ^(§)The structures of the latanoprost isomers are set out in FIG. 2.

-   -   No isomers were detected after 11 months storage at freezer         temperature when the purified Latanoprost was analyzed with a         typical HPLC method for isomer content determination to the         following detection levels:

Isomer Detection Limit C11-beta 0.03% C15R-Trans 0.03% C15S-Trans 0.05% C15S-Cis 0.03% Latanoprost 0.03% 

What is claimed:
 1. A latanoprost composition comprising C11-beta isomer of latanoprost in an amount greater than 0% and less than 0.03%, and containing less than 0.03% C15R-Trans isomer of latanoprost, less than 0.05% C15S-Trans isomer of latanoprost, and less than 0.03% C15S-Cis isomer of latanoprost.
 2. A latanoprost composition comprising C11-beta isomer of latanoprost in an amount greater than 0% and less than 0.03%, and containing less than 0.03% C15R-Trans isomer of latanoprost, less than 0.05% C15S-Trans isomer of latanoprost, and less than 0.03% C15S-Cis isomer of latanoprost, after 11 months storage at freezer temperature.
 3. C11-beta isomer of latanoprost.
 4. A latanoprost composition comprising C11-beta isomer of latanoprost in an amount greater than 0% and less than 0.03%.
 5. A latanoprost composition comprising at C11-beta isomer of latanoprost in an amount greater than 0% and less than 0.03%, after 11 months storage at freezer temperature.
 6. A composition according to claim 1, wherein the latanopost composition is at least 99% chemically pure.
 7. A composition according to claim 1, wherein the latanopost composition is at least 99.5% chemically pure.
 8. A composition according to claim 1, wherein the latanopost composition is at least 99.8% chemically pure.
 9. A composition according to claim 1, wherein the latanopost composition is at least 99% isometrically pure.
 10. A composition according to claim 1, wherein the latanopost composition is at least 99.5% isometrically pure.
 11. A composition according to claim 1, wherein the latanopost composition is at least 99.8% isometrically pure.
 12. A composition according to claim 4, wherein the latanopost composition is at least 99% chemically pure.
 13. A composition according to claim 4, wherein the latanopost composition is at least 99.5% chemically pure.
 14. A composition according to claim 4, wherein the latanopost composition is at least 99.8% chemically pure.
 15. A composition according to claim 4, wherein the latanopost composition is at least 99% isometrically pure.
 16. A composition according to claim 4, wherein the latanopost composition is at least 99.5% isometrically pure.
 17. A composition according to claim 4, wherein the latanopost composition is at least 99.8% isometrically pure. 